The control of bleeding and clotting are extremely important in medical and surgical procedures. Protein C (PC), a vitamin-K-dependent plasma glycoprotein, is of major physiological importance in the control of bleeding. Protein C exists in two forms, an inactive or nascent form and an active form. Inactive protein C is composed of two polypeptide chains with a molecular weight of 62 kDa. It is present as a serine proteinase zymogen whose limited proteolysis results in the release of a dodecapeptide from the N-terminus of the heavy chain and the conversion of the zymogen form to activated PC (APC). PC activation is normally catalyzed by its interaction with a 1:1 stoichiometric complex of thrombin with thrombomodulin, a protein expressed on endothelial cells under physiological conditions.
APC is a natural anticoagulant that degrades the activated forms of the clotting cofactors V and VIII (i.e., Va and VIIIa), thereby terminating the procoagulant activity of Factors Va and VIlla, in the presence of phospholipids and a cofactor, protein S. Thus, APC negatively regulates the blood coagulation system. The thrombotic tendency of patients, as represented by a tendency for blood clot formation, with either a PC-deficiency or genetic abnormality clearly demonstrates the importance of the PC pathway in the control of coagulation.
Decreased plasma levels of PC have been reported in patients with disseminated intravascular coagulation (DIC) or in sepsis (i.e., the presence in the blood or other tissues of pathogenic microorganisms or their toxins, also referred to as septicemia), which precludes DIC. In parallel with this effect, increased levels of APC complexed with 1-proteinase inhibitor or APC inhibitor have been observed in septic patients, indicating consumption of the active enzyme. Recent studies have shown that monocyte derived tissue factor initiates the extrinsic coagulation pathway in sepsis or DIC. Accordingly, any thrombin generated would form a complex with thrombomodulin and activate PC, resulting in a significant decrease in concentration of this anticoagulant factor. Since endotoxin and/or endotoxin-induced tumor necrosis factor can stimulate monocytes to express tissue factor, the tendency has been to recognize lipopolysaccharide as the primary bacterial agent responsible for decreased PC levels in sepsis. Besides endotoxin, however, bacteria release a variety of proteinases, some of which are known to activate coagulation factors, including prothrombin, factor XII and prekallikrein.
The present invention provides methods for controlling blood coagulation, and suitable pharmaceutical compositions.
In one embodiment is a method for producing activated protein C in a medium by contacting the medium with an effective amount of an arginine-specific cysteine proteinase of bacterial origin.
In a second embodiment is a method for producing activated protein C in blood by contacting the blood with an effective amount of a polypeptide represented by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, active fragments or active modifications thereof.
In a third embodiment is a method for producing activated protein C in blood by contacting the blood with an effective amount of a polypeptide encoded by a polynucleotide, the complement of which hybridizes to nucleotides encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 under standard hybridization conditions.
In a fourth embodiment is a method of inhibiting blood coagulation in a patient by administering an effective anticoagulant amount of an argimne-specific cysteine proteinase of bacterial origin.
In a fifth embodiment is a method of inhibiting blood coagulation in a patient by administering an effective anticoagulant amount of a polypeptide represented by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, active fragments or active modifications thereof.
In a sixth embodiment is a method of inhibiting blood coagulation in a patient by administering an effective anticoagulant amount of a polypeptide encoded by a polynucleotide, the complement of which hybridizes to nucleotides encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 under standard hybridization conditions.
In a seventh embodiment is an anticoagulant composition that includes an effective amount of an arginine-specific cysteine proteinase of bacterial origin, or salt thereof, and a pharmaceutically acceptable carrier.
In an eighth embodiment is an anticoagulant composition that includes an effective amount of a polypeptide represented by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, active fragments or active modifications thereof.
In a ninth embodiment is an anticoagulant composition that includes an effective amount of a polypeptide encoded by a polynucleotide, the complement of which hybridizes to nucleotides encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 under standard hybridization conditions.
In a tenth embodiment is a method for reversing anticoagulation in a medium by contacting the medium with an inhibitor of an arginine-specific cysteine proteinase of bacterial origin.
In an eleventh embodiment is an anticoagulant reversing composition that includes an effective amount of an inhibitor of an arginine-specific cysteine proteinase of bacterial origin, or salt thereof, and a pharmaceutically acceptable carrier.
In a twelfth embodiment is a method for treating septicemia in a patient by administering an effective amount of an inhibitor of an arginine-specific cysteine proteinase of bacterial origin, or salt thereof. Preferably, the septicemia is induced by pathogenic bacteria.
In yet another embodiment is a method for treating disseminated intravascular coagulation in a patient by administering an effective amount of an inhibitor of an arginine-specific cysteine proteinase of bacterial origin, or salt thereof. Preferably, the disseminated intravascular coagulation is induced by pathogenic bacteria.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. xe2x80x9cPeptidase,xe2x80x9d xe2x80x9cproteinase,xe2x80x9d and xe2x80x9cproteasexe2x80x9d are used interchangeably to refer to enzymes and are encompassed within the definition of polypeptide.
The term xe2x80x9cisolatedxe2x80x9d means that a polypeptide is either removed from its natural environment or synthetically derived. Preferably, the polypeptide is purified, i.e., essentially free from any other polypeptides and associated cellular products or other impurities.
The terms xe2x80x9chybridizes,xe2x80x9d xe2x80x9chybridizing,xe2x80x9d and xe2x80x9chybridizationxe2x80x9d mean that a single stranded polynucleotide forms a noncovalent interaction with a complementary polynucleotide under certain conditions, as described herein.